Production of compounds of pharmaceutical significance by culturing bacteria and isolation of products produced in culture is an expanding field of science. An impediment to obtaining regulatory approval for products produced in this manner is encountered whenever the bacterial organism being cultured requires complex animal components for its growth. For example, culture of Streptococcus pneumoniae (pneumococci), to produce pneumococal polysaccharides for vaccine production, particularly at the inoculum development stage, is typically performed in media containing complex bovine or ovine derived products such as peptones, gelatins, caseins, or blood derived products. S. pneumoniae are known to be fastidious, having nutritional requirements for choline [Rave et al., J. Bact., 40, 695-704 (1940)], vitamins [Adams et al., J. Bact., 49 401-409 (1945)], and biotin. [Bohonos and Sabborow, Arch. Biochem. 3, 257-259 (1943)]. Even where subsequent culture of bacteria in media free of complex bovine or ovine derived products has been achieved, initial development of the culture inoculum of S. pneumoniae has been accomplished in blood-containing or bovine/ovine derived product media. This invention demonstrates the feasability of inoculum development in a medium devoid of these complex and potentially troublesome components.
One commercially available medium useful for bacterial culture is known as PYG. PYG contains:
______________________________________ peptone 5 g trypticase 5 g yeast extract 10 g resazurin solution 4 ml salts solution 40 ml distilled water 1000 ml hemin solution 10 ml vitamin K 0.2 ml cysteine HCl.H.sub.2 O 0.5 g glucose 10 g ______________________________________
(see Anaerobe Laboratory Manual, 4th Edition, 1977, Holdeman et al. eds; Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061, p13). S. pneumoniae can be cultured in this medium, but since the medium contains beef peptone, it is not desirable for production of vaccine components.
One significant reason for using a culture medium free of animal or blood products is to reduce or avoid the possibility of contamination by agents which cause spongiform encephalopathies, such as Prion Disease [see, for example, The Lancet, vol. 336, p21-22, Jul. 7, 1990, and references cited therein]. The etiologic agent of such diseases as scrapie in animals, Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, and kuru in man, is thought to be transimissible by animal blood products, particularly bovine or ovine derived products.
We have discovered a culture medium completely devoid of complex animal derivatives which nonetheless supports the growth of the pneumococci, such as Streptococcus pneumoniae strains (Danish nomenclature based on serotype) 1, 2, 3, 4, 5, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F. The medium does not contain bovine or ovine-derived products, nor does it contain blood. Furthermore, the inocula derived in this new medium can be used to initiate production of the pneumococcal capsular polysaccharide which can be harvested and used to prepare vaccines, as described for example in U.S. Pat. No. 4,695,624.
The advantage of this new medium is the elimination of animal components (especially bovine or ovine derived products) from the product production process. By eliminating animal components, the dangers of contamination with reactive blood group substances or adventitious agents are reduced, and the need to remove these contaminants during purification is eliminated. It also becomes less necessary to demonstrate the absence of such contaminants in the final vaccine product.
Thus, according to this invention, a new medium composition for culturing bacteria is provided. A method for using said composition, particularly for inoculum development, is also provided. The invention will allow production of fastidious bacteria such as S. pneumoniae, and frozen seed stocks thereof which have not had contact with bovine or ovine derived materials. This will facilitate the regulatory review process for the pharmaceutical products derived from culture of such organisms. Thus, according to this invention, culture growth and inoculum development can be obtained in a medium free of bovine or ovine derived products. Furthermore, the invention provides an alternate and preferred culture medium and method for production of inocula for pneumococcal capsular polysaccharide production.